Natural Killer Cells Control Tumor Growth by Sensing a Growth Factor.

Many tumors produce platelet-derived growth factor (PDGF)-DD, which promotes cellular proliferation, epithelial-mesenchymal transition, stromal reaction, and angiogenesis through autocrine and paracrine PDGFRß signaling. By screening a secretome library, we found that the human immunoreceptor NKp44, encoded by NCR2 and expressed on natural killer (NK) cells and innate lymphoid cells, recognizes PDGF-DD. PDGF-DD engagement of NKp44 triggered NK cell secretion of interferon gamma (IFN)-γ and tumor necrosis factor alpha (TNF-α) that induced tumor cell growth arrest. A distinctive transcriptional signature of PDGF-DD-induced cytokines and the downregulation of tumor cell-cycle genes correlated with NCR2 expression and greater survival in glioblastoma. NKp44 expression in mouse NK cells controlled the dissemination of tumors expressing PDGF-DD more effectively than control mice, an effect enhanced by blockade of the inhibitory receptor CD96 or CpG-oligonucleotide treatment. Thus, while cancer cell production of PDGF-DD supports tumor growth and stromal reaction, it concomitantly activates innate immune responses to tumor expansion.

Endothelial PDGF-D contributes to neurovascular protection after ischemic stroke by rescuing pericyte functions.

Ischemic stroke induces neovascularization of the injured tissue as an attempt to promote structural repair and neurological recovery. Angiogenesis is regulated by pericytes that potently react to ischemic stroke stressors, ranging from death to dysfunction. Platelet-derived growth factor (PDGF) receptor (PDGFR)ß controls pericyte survival, migration, and interaction with brain endothelial cells. PDGF-D a specific ligand of PDGFRß is expressed in the brain, yet its regulation and role in ischemic stroke pathobiology remains unexplored. Using experimental ischemic stroke mouse model, we found that PDGF-D is transiently induced in brain endothelial cells at the injury site in the subacute phase. To investigate the biological significance of PDGF-D post-ischemic stroke regulation, its subacute expression was either downregulated using siRNA or upregulated using an active recombinant form. Attenuation of PDGF-D subacute induction exacerbates neuronal loss, impairs microvascular density, alters vascular permeability, and increases microvascular stalling. Increasing PDGF-D subacute bioavailability rescues neuronal survival and improves neurological recovery. PDGF-D subacute enhanced bioavailability promotes stable neovascularization of the injured tissue and improves brain perfusion. Notably, PDGF-D enhanced bioavailability improves pericyte association with brain endothelial cells. Cell-based assays using human brain pericyte and brain endothelial cells exposed to ischemia-like conditions were applied to investigate the underlying mechanisms. PDGF-D stimulation attenuates pericyte loss and fibrotic transition, while increasing the secretion of pro-angiogenic and vascular protective factors. Moreover, PDGF-D stimulates pericyte migration required for optimal endothelial coverage and promotes angiogenesis. Our study unravels new insights into PDGF-D contribution to neurovascular protection after ischemic stroke by rescuing the functions of pericytes.

PDGF-D-PDGFRß signaling enhances IL-15-mediated human natural killer cell survival.

The axis of platelet-derived growth factor (PDGF) and PDGF receptor-beta (PDGFRß) plays prominent roles in cell growth and motility. In addition, PDGF-D enhances human natural killer (NK) cell effector functions when binding to the NKp44 receptor. Here, we report an additional but previously unknown role of PDGF-D, whereby it mediates interleukin-15 (IL-15)-induced human NK cell survival but not effector functions via its binding to PDGFRß but independent of its binding to NKp44. Resting NK cells express no PDGFRß and only a low level of PDGF-D, but both are significantly up-regulated by IL-15, via the nuclear factor κB signaling pathway, to promote cell survival in an autocrine manner. Both ectopic and IL-15-induced expression of PDGFRß improves NK cell survival in response to treatment with PDGF-D. Our results suggest that the PDGF-D-PDGFRß signaling pathway is a mechanism by which IL-15 selectively regulates the survival of human NK cells without modulating their effector functions.

Combination therapy with chitosan/siRNA nanoplexes targeting PDGF-D and PDGFR-ß reveals anticancer effect in breast cancer.

BACKGROUND: Platelet derived growth factors (PDGF)-D and the expression of its receptor increase in neoplastic progression of cancer. Co-silencing of growth factor and receptor can be suggested as an important strategy for effective cancer therapy. In the present study, we hypothesized that suppression of PDGF-D signaling pathway with small interfering RNAs (siRNAs) targeting both PDGF-D and PDGF receptor (PDGFR)-ß is a promising strategy for anticancer therapy. METHODS: Chitosan nanoplexes containing dual and single siRNA were prepared at different weight ratios and controlled by gel retardation assay. Characterization, cellular uptake, gene silencing and invasion studies were performed. The effect of nanoplexes on breast tumor growth, PDGF expression and apoptosis was investigated. RESULTS: We have shown that downregulation of PDGF-D and PDGFR-ß with chitosan/siRNA nanoplex formulations reduced proliferation and invasion in breast cancer cells. In the in vivo breast tumor model, it was determined that the intratumoral administration of chitosan/siPDGF-D/siPDGFR-ß nanoplexes markedly decreased the tumor volume and PDGF-D and PDGFR-ß mRNA and protein expression levels and increased apoptosis. CONCLUSIONS: According to the results obtained, we evaluated the effect of PDGF-D and PDGFR-ß on breast tumor development and showed that RNAi-mediated inhibition of this pathway formulated with chitosan nanoplexes can be considered as a new breast cancer therapy strategy.

Positive regulatory loop of platelet-derived growth factor DD-induced STAT3 activation is associated with poor prognosis in advanced urothelial carcinoma.

Immune checkpoint inhibitor (ICI) therapy has been established for patients with advanced urothelial cancer (UC). The necessity of overcoming resistance to ICIs and identifying a predictive factor for the same has been highlighted, such as the assessment of combination therapy with other targeted drugs and the characterization of molecular signatures in the tumor microenvironment. Recently, we reported that low hemoglobin (Hb) levels and a high platelet-to-lymphocyte ratio (PLR) were significantly associated with overall survival in patients with UC who did not benefit from pembrolizumab treatment. In the present study, we identified a possible link between these unfavorable prognostic indicators and PDGF-DD-induced STAT3 activation in UC. Overlapping patients between the high STAT3- or phosphorylated STAT3-positive score group (as assessed by immunohistochemistry) and low Hb levels or high PLR group (as assessed by blood tests) showed significantly worse outcomes after pembrolizumab treatment. Additionally, using the bladder cancer JMSU1 cell line, we demonstrated a possible positive regulatory loop between autocrine/paracrine PDGF-DD and STAT3 signaling. Therefore, we suggest that STAT3 inhibition and PDGF-DD detection in the tumor microenvironment might represent a potential therapeutic strategy to overcome resistance to pembrolizumab. Moreover, this can help identify patients with UC who could benefit from combination treatment.

Gene fusions in superficial mesenchymal neoplasms: Emerging entities and useful diagnostic adjuncts.

Cutaneous mesenchymal neoplasms are diagnostically challenging because of their overlapping morphology, and, often, the limited tissue in skin biopsy specimens. Molecular and cytogenetic techniques have identified characteristic gene fusions in many of these tumor types, findings that have expanded our understanding of disease pathogenesis and motivated development of useful ancillary diagnostic tools. Here, we provide an update of new findings in tumor types that can occur in the skin and superficial subcutis, including dermatofibrosarcoma protuberans, benign fibrous histiocytoma, epithelioid fibrous histiocytoma, angiomatoid fibrous histiocytoma, glomus tumor, myopericytoma/myofibroma, non-neural granular cell tumor, CIC-rearranged sarcoma, hybrid schwannoma/perineurioma, and clear cell sarcoma. We also discuss recently described and emerging tumor types that can occur in superficial locations and that harbor gene fusions, including nested glomoid neoplasm with GLI1 alterations, clear cell tumor with melanocytic differentiation and ACTIN::MITF translocation, melanocytic tumor with CRTC1::TRIM11 fusion, EWSR1::SMAD3-rearranged fibroblastic tumor, PLAG1-rearranged fibroblastic tumor, and superficial ALK-rearranged myxoid spindle cell neoplasm. When possible, we discuss how fusion events mediate the pathogenesis of these tumor types, and we also discuss the related diagnostic and therapeutic implications of these events.

MicroRNA miR-145-5p Inhibits Cutaneous Wound Healing by Targeting PDGFD in Diabetic Foot Ulcer.

Diabetic foot ulcer (DFU) is one major, common and serious chronic complication of diabetes mellitus, which is characterized by high incidence, high risk, high burden, and high treatment difficulty and is a leading cause of disability and death in patients with diabetes. Long-term hyperglycemia can result in cellular dysfunction of fibroblasts, which play pivotal roles in wound healing. MicroRNAs (miRNAs) were reported to mediate the pathological processes of multiple diseases, including diabetic wound healing. This research aimed to investigate the functional role of miR-145-5p in high-glucose (HG)-exposed fibroblasts and in DFU mouse models. Human foreskin fibroblast cells (HFF-1) were stimulated by HG to induce cell injury. MiR-145-5p level in HG-stimulated HFF-1 cells was detected via RT-qPCR. The binding between miR-145-5p and PDGFD was validated by Luciferase reporter assay. The effects of the miR-145-5p/PDGFD axis on the viability, migration, and apoptosis of HG-exposed HFF-1 cells were determined by CCK-8, wound healing, and flow cytometry assays. DFU mouse models were subcutaneously injected at the wound edges with miR-145-5p inhibitor/mimics. Images of the wounds were captured on day 0 and 8 post-injection, and wound samples were collected after mice were sacrificed for histological analysis by H&E staining. HG decreased cell viability and increased miR-145-5p expression in HFF-1 cells in a dose- and time-dependent manner. MiR-145-5p downregulation promoted cell viability and migration and inhibited cell apoptosis of HG-stimulated HFF-1 cells, while miR-145-5p overexpression exerted an opposite effect on cell viability, migration, and apoptosis. PDGFD was a direct target gene of miR-145-5p, whose silencing reversed the influence of miR-145-5p downregulation on HG-induced cellular dysfunction of HFF-1 cells. Additionally, downregulating miR-145-5p facilitated while overexpressing miR-145-5p inhibited wound healing in DFU mouse models. MiR-145-5p level was negatively associated with PDGFD level in wound tissue samples of DFU mouse models. MiR-145-5p inhibition improves wound healing in DFU through upregulating PDGFD expression.

PDGFD switches on stem cell endothelial commitment.

The critical factors regulating stem cell endothelial commitment and renewal remain not well understood. Here, using loss- and gain-of-function assays together with bioinformatic analysis and multiple model systems, we show that PDGFD is an essential factor that switches on endothelial commitment of embryonic stem cells (ESCs). PDGFD genetic deletion or knockdown inhibits ESC differentiation into EC lineage and increases ESC self-renewal, and PDGFD overexpression activates ESC differentiation towards ECs. RNA sequencing reveals a critical requirement of PDGFD for the expression of vascular-differentiation related genes in ESCs. Importantly, PDGFD genetic deletion or knockdown increases ESC self-renewal and decreases blood vessel densities in both embryonic and neonatal mice and in teratomas. Mechanistically, we reveal that PDGFD fulfills this function via the MAPK/ERK pathway. Our findings provide new insight of PDGFD as a novel regulator of ESC fate determination, and suggest therapeutic implications of modulating PDGFD activity in stem cell therapy.

Dermatofibrosarcoma protuberans with platelet-derived growth factor-D rearrangement; two cases with morphologically distinct presentations.

Dermatofibrosarcoma protuberans (DFSP) is a mesenchymal neoplasm that is usually located in the dermis or subcutis and is locally aggressive. Rarely, these lesions may undergo fibrosarcomatous transformation, which is thought to increase their metastatic potential. DFSP is classically associated with a 17;22 translocation (or ring chromosome thereof) resulting in fusion of the COL1A1 and PDGFB genes. However, variant fusions involving PDGFD have been recently reported. Herein, we present two morphologically diverse cases of DFSP with PDGFD rearrangement. Case 1 is a 68-year-old female with a left dorsal foot lesion. Morphologically, the lesion is unusual as it is a well-circumscribed, hypercellular, subcutaneous nodule with uniform CD34-positive spindle cells arranged in a herringbone pattern without storiform arrangement or "honeycombing" fat entrapment. It was diagnosed as pure fibrosarcomatous DFSP. Case 2 is a 37-year-old male with a right supra-auricular lesion. Morphologically, the lesion displays classic DFSP features including bland CD34-positive spindle cells with storiform growth, fat entrapment, and infiltrative borders. Both lesions were negative for COL1A1-PDGFB fusion but positive for PDGFD rearrangement by fluorescence in situ hybridization (FISH) analysis. FISH testing for PDGFD rearrangement should be performed in cases where there is a high suspicion for DFSP but initial studies for COL1A1-PDGFB are negative.

GLIS1, a potential candidate gene affect fat deposition in sheep tail.

Fat deposition in sheep tails is as a result of a complicated mechanism. Mongolian sheep (MG) and Small Tail Han sheep (STH) are two fat-tailed Chinese indigenous sheep breeds while DairyMeade and East Friesian (DS) are two thin-tailed dairy sheep breeds recently introduced to China. In this study, population genomics analysis was applied to identify candidate genes associated with sheep tails based on an in-depth whole-genome sequencing of MG, STH and DS. The selective signature analysis demonstrated that GLIS1, LOC101117953, PDGFD and T were in the significant divergent regions between DS and STH-MG. A nonsynonymous point mutation (g.27807636G>T) was found within GLIS1 in STH-MG and resulted in a Pro to Thr substitution. As a pro-adipogenic factor, GLIS1 may play critical roles in the mesodermal cell differentiation during fetal development affecting fat deposition in sheep tails. This study gives a new insight into the genetic basis of species-specific traits of sheep tails.

Genomic analysis of worldwide sheep breeds reveals PDGFD as a major target of fat-tail selection in sheep.

BACKGROUND: Fat tail is a unique trait in sheep acquired during domestication. Several genomic analyses have been conducted in sheep breeds from limited geographic origins to identify the genetic factors underlying this trait. Nevertheless, these studies obtained different candidates. The results of these regional studies were easily biased by the breed structures. RESULTS: To minimize the bias and distinguish the true candidates, we used an extended data set of 968 sheep representing 18 fat-tailed breeds and 14 thin-tailed breeds from around the world, and integrated two statistical tests to detect selection signatures, including Genetic Fixation Index (FST) and difference of derived allele frequency (ΔDAF). The results showed that platelet derived growth factor D (PDGFD) exhibited the highest genetic differentiation between fat- and thin-tailed sheep breeds. Analysis of sequence variation identified that a 6.8-kb region within the first intron of PDGFD is likely the target of positive selection and contains regulatory mutation(s) in fat-tailed sheep. Histological and gene expression analyses demonstrated that PDGFD expression is associated with maturation and hemostasis of adipocytes. Further retrospective analysis of public transcriptomic datasets revealed that PDGFD expression is down-regulated during adipogenesis in both human and mouse, and is higher in fat tissues of obese individuals than that in lean individuals. CONCLUSIONS: These results reveal that PDGFD is the predominant factor for the fat tail phenotype in sheep by contributing to adiopogenesis and maintaining the hemostasis of mature adipocytes. This study provides insights into the selection of fat-tailed sheep and has important application to animal breeding, as well as obesity-related human diseases.

Platelet-derived growth factor-D expression mediates the effect of differentiated degree on prognosis in epithelial ovarian cancer.

Platelet-derived growth factor-D (PDGF-D) can enhance invasion and metastasis in several human malignancies. Although several studies have been performed to investigate the association between clinicopathological characteristics and prognosis in epithelial ovarian cancer (EOC), the mediation effect of PDGF-D on above-mentioned association have been seldom assessed. In this study, we detected the PDGF-D expression from the tissues of patients with EOC and further collected clinicopathological characteristics and prognostic information to identify whether PDGF-D mediated the effect of differentiated degree on prognosis in patients with EOC. A total of 190 paraffin-embedded tissue samples from patients with EOC between July 2005 and December 2010 were collected. We performed a Kaplan-Meier analysis for the association between differentiated degree and prognosis followed by a causal mediation analysis. The analysis results indicated that differentiated degree was associated with prognosis and PDGF-D mediated the effect of differentiated degree on prognosis in patients with EOC, which might be a potential target for ovarian cancer treatment.

Neuropilin 1 binds PDGF-D and is a co-receptor in PDGF-D-PDGFRß signaling.

Platelet-derived growth factor (PDGF)-D is a PDGF receptor ß (PDGFRß)-specific ligand implicated in a number of pathological conditions, such as cardiovascular disease and cancer, but its biological function remains incompletely understood. In this study, we demonstrate that PDGF-D binds directly to neuropilin 1 (NRP1), in a manner that requires the PDGF-D C-terminal Arg residue. Stimulation with PDGF-D, but not PDGF-B, induced PDGFRß-NRP1 complex formation in fibroblasts. Additionally, PDGF-D induced translocation of NRP1 to cell-cell junctions in endothelial cells, independently of PDGFRß, altering the availability of NRP1 for VEGF-A-VEGFR2 signaling. PDGF-D showed differential effects on pericyte behavior in ex vivo sprouting assays compared to PDGF-B. Furthermore, PDGF-D-induced PDGFRß-NRP1 interaction can occur in trans between molecules located in different cells (endothelial cells and pericytes). In summary, we show that NRP1 can act as a co-receptor for PDGF-D-PDGFRß signaling and is possibly implicated in intercellular communication in the vascular wall.

Possible Role and Therapeutic Target of PDGF-D Signalling in Colorectal Cancer.

Platelet-derived growth factor D (PDGF-D) has been shown to mediate cellular processes of importance in cancer progression. This study aimed to investigate the expression and putative involvement of PDGF-D signaling in colorectal carcinogenesis. PDGF-D was expressed in vascular endothelial cells in tumor and normal tissues. PDGF-D stimulation of cells altered genes of importance in carcinogenic processes. In addition, PDGF-D increased the proliferation rate while imatinib inhibited these effects. PDGF-D and its PDGF receptor beta (PDGFR-ß) are expressed in colorectal cancer and blockage of PDGF-D/PDGFR-ß signaling using tyrosine kinase inhibitors, such as imatinib, might be important in inhibiting tumor-promoting actions.

Dermatofibrosarcoma protuberans with a novel COL6A3-PDGFD fusion gene and apparent predilection for breast.

Dermatofibrosarcoma protuberans is a locally aggressive superficial mesenchymal neoplasm. It typically occurs in adulthood, and has been reported to have a slight male predilection. Tumors have a characteristic histopathologic appearance, including: storiform architecture, infiltrative "honeycomb" growth within subcutaneous adipose tissue, and immunoreactivity for CD34. Virtually all molecularly characterized cases to date have been found to harbor a COL1A1-PDGFB fusion product. Following identification of an index patient with a novel COL6A3-PDGFD fusion gene, we undertook a molecular investigation, using a combination of RNA sequencing and fluorescence in situ hybridization (FISH), to assess the prevalence of PDGFD rearrangement in dermatofibrosarcoma protuberans (N = 63). Three additional patients were found to have balanced PDGFD rearrangements. Interestingly, all 4 tumors arose on the breast of females. As a result, we subsequently examined 16 additional cases of primary breast dermatofibrosarcoma protuberans, identifying 2 additional tumors with PDGFD rearrangement. The morphology and immunophenotype of all 6 cases was analogous to those with the canonical COL1A1-PDGFB fusion; none of the cases showed fibrosarcomatous transformation. This study illustrates that the COL6A3-PDGFD fusion product is rare in dermatofibrosarcoma protuberans, and associated with an apparent predilection for breast. An awareness of this variant is important for pathologists, as it will not be detected using conventional reverse transcription polymerase chain reaction or FISH-based diagnostic assays for dermatofibrosarcoma protuberans.

Matriptase activation and shedding through PDGF-D-mediated extracellular acidosis.

Activation of ß-platelet-derived growth factor receptor (ß-PDGFR) is associated with prostate cancer (PCa) progression and recurrence after prostatectomy. Analysis of the ß-PDGFR ligands in PCa revealed association between PDGF-D expression and Gleason score as well as tumor stage. During the course of studying the functional consequences of PDGF ligand-specific ß-PDGFR signaling in PCa, we discovered a novel function of PDGF-D for activation/shedding of the serine protease matriptase leading to cell invasion, migration, and tumorigenesis. The present study showed that PDGF-D, not PDGF-B, induces extracellular acidification, which correlates with increased matriptase activation. A cDNA microarray analysis revealed that PDGF-D/ß-PDGFR signaling upregulates expression of the acidosis regulator carbonic anhydrase IX (CAIX), a classic target of the transcriptional factor hypoxia-inducible factor-1α (HIF-1α). Cellular fractionation displayed a strong HIF-1α nuclear localization in PDGF-D-expressing cells. Treatment of vector control or PDGF-B-expressing cells with the HIF-1α activator CoCl2 led to increased CAIX expression accompanied by extracellular acidosis and matriptase activation. Furthermore, the analysis of the CAFTD cell lines, variants of the BPH-1 transformation model, showed that increased PDGF-D expression is associated with enhanced HIF-1α activity, CAIX induction, cellular acidosis, and matriptase shedding. Importantly, shRNA-mediated knockdown of CAIX expression effectively reversed extracellular acidosis and matriptase activation in PDGF-D-transfected BPH-1 cells and in CAFTD variants that express endogenous PDGF-D at a high level. Taken together, these novel findings reveal a new paradigm in matriptase activation involving PDGF-D-specific signal transduction leading to extracellular acidosis.

Accelerated fracture healing in transgenic mice overexpressing an anabolic isoform of fibroblast growth factor 2.

The effect of targeted expression of an anabolic isoform of basic fibroblast growth factor (FGF2) in osteoblastic lineage on tibial fracture healing was assessed in mice. Closed fracture of the tibiae was performed in Col3.6-18 kDaFgf2-IRES-GFPsaph mice in which a 3.6 kb fragment of type I collagen promoter (Col3.6) drives the expression of only the 18 kD isoform of FGF2 (18 kDaFgf2/LMW) with green fluorescent protein-sapphire (GFPsaph) as well as Vector mice (Col3.6-IRES-GFPsaph, Vector) that did not harbor the FGF2 transgene. Radiographic, micro-CT, DEXA, and histologic analysis of fracture healing of tibiae harvested at 3, 10 and 20 days showed a smaller fracture callus but accelerated fracture healing in LMWTg compared with Vector mice. At post fracture day 3, FGF receptor 3 and Sox 9 mRNA were significantly increased in LMWTg compared with Vector. Accelerated fracture healing was associated with higher FGF receptor 1, platelet derived growth factors B, C, and D, type X collagen, vascular endothelial cell growth factor, matrix metalloproteinase 9, tartrate resistant acid phosphatase, cathepsin K, runt-related transcription factor-2, Osterix and Osteocalcin and lower Sox9, and type II collagen expression at 10 days post fracture. We postulate that overexpression of LMW FGF2 accelerated the fracture healing process due to its effects on factors that are important in chondrocyte and osteoblast differentiation and vascular invasion.

PDGF-D gene polymorphism is associated with increased cardiovascular mortality in elderly men.

BACKGROUND: Platelet-derived growth factor (PDGF) D has been reported to be active in fibroblasts, and in areas of myocardial infarction. In this longitudinal study we evaluated the association between PDGF-D polymorphism and cardiovascular mortality, and attempted to discover whether specific genotype differences regarding risk could be observed, and if gender differences could be seen. METHODS: Four hundred seventy-six elderly community participants were included in this study. All participants underwent a clinical examination, echocardiography, and blood sampling including PDGF-D single nucleotide polymorphism (SNP) analyses of the rs974819 A/A, G/A and G/G SNP. The follow-up time was 6.7 years. RESULTS: No specific genotype of rs974819 demonstrated increased cardiovascular mortality in the total population, however, the male group with genotypes A/A and G/A demonstrated an increased risk that persisted in a multivariate evaluation where adjustments were made for well-known cardiovascular risk factors (2.7 fold compared with the G/G genotype). No corresponding finding was observed in the female group. CONCLUSION: We report here for the first time that the genotypes G/A or A/A of the SNP rs974819 near PDGF-D exhibited a 2.7 fold increased cardiovascular mortality risk in males. Corresponding increased risk could not be observed in either the total population and thus not in the female group. However, the sample size is was small and the results should be regarded as hypothesis-generating, and thus more research in the field is recommended.

PDGF-D promotes dermal fibroblast invasion in 3-dimensional extracellular matrix via Snail-mediated MT1-MMP upregulation.

Increasing attention has been focused on the malignant tumor microenvironment, which plays important roles in tumor occurrence, progression and metastasis. Fibroblasts are recruited by platelet-derived growth factor (PDGFs) and invade the tumor microenvironment. In the PDGF family, PDGF-B has been reported to play an important role in the recruitment and invasion programs. However, whether PDGF-D plays a role in these programs remains unclear. We generated a recombinant plasmid expressing human PDGF-D and transfected the plasmid to dermal fibroblasts to examine the effects on cell invasive activities in 3D type I collagen gels. PDGF-D plasmid transfection enhanced fibroblast invasive activities both in invasive cell numbers and invasion depth in 3D collagen gels. These effects were blocked by Snail-specific siRNA transfection. PDGF-D transfection significantly induced Snail expression at both mRNA and protein levels. PDGF-D further upregulated MT1-MMP mRNA and protein expressions and this was inhibited when Snail was knocked down by siRNA. Both Snail and MT1-MMP expressions in fibroblasts and cellular invasive activities in 3D collagen induced by PDGF-D were inhibited by LY294002, SP600125, and U1026, the inhibitors of PI3K, JNK, and ERK1/2 signaling pathways, respectively. However, no effects were observed in response to the P38MAPK signaling pathway inhibitor SB203580. These effects of PDGF-D were confirmed by using the culture supernatants of the transfectants. Taken together, these data demonstrate that PDGF-D plays important roles in the recruitment and invasion programs of fibroblasts via the activation of PI3K, JNK and ERK1/2 signaling pathways, and upregulation of Snail and downstream effecter MT1-MMP. These findings indicate that PDGF-D is an important player in the tumor microenvironment for fibroblast recruitment.

Functional regulation of adipose-derived stem cells by PDGF-D.

Platelet-derived growth factor-D (PDGF-D) was recently identified, and acts as potent mitogen for mesenchymal cells. PDGF-D also induces cellular transformation and promotes tumor growth. However, the functional role of PDGF-D in adipose-derived stem cells (ASCs) has not been identified. Therefore, we primarily investigated the autocrine and paracrine roles of PDGF-D in this study. Furthermore, we identified the signaling pathways and the molecular mechanisms involved in PDGF-D-induced stimulation of ASCs. It is of interest that PDGF-B is not expressed, but PDGF-D and PDGF receptor-ß are expressed in ASCs. PDGF-D showed the strongest mitogenic effect on ASCs, and PDGF-D regulates the proliferation and migration of ASCs through the PI3K/Akt pathways. PDGF-D also increases the proliferation and migration of ASCs through generation of mitochondrial reactive oxygen species (mtROS) and mitochondrial fission. mtROS generation and fission were mediated by p66Shc phosphorylation, and BCL2-related protein A1 and Serpine peptidase inhibitor, clade E, member 1 mediated the proliferation and migration of ASCs. In addition, PDGF-D upregulated the mRNA expression of diverse growth factors such as vascular endothelial growth factor A, fibroblast growth factor 1 (FGF1), FGF5, leukemia inhibitory factor, inhibin, beta A, interleukin 11, and heparin-binding EGF-like growth factor. Therefore, the preconditioning of PDGF-D enhanced the hair-regenerative potential of ASCs. PDGF-D-induced growth factor expression was attenuated by a pharmacological inhibitor of mitogen-activated protein kinase pathway. In summary, PDGF-D is highly expressed by ASCs, where it acts as a potent mitogenic factor. PDGF-D also upregulates growth factor expression in ASCs. Therefore, PDGF-D can be considered a novel ASC stimulator, and used as a preconditioning agent before ASC transplantation.